Project 1. The pedunculopontine (PPTg) and laterodorsal tegmental nuclei (LDTg) provide cholinergic, GABAergic and glutamatergic afferents to the ventral tegmental area (VTA). We recently demonstrated that the PPTg and the LDTg contain independent populations of cholinergic, glutamatergic and GABAergic neurons (Wang & Morales, EJN, 2009). Here, by combining retrograde tract tracing and in situ hybridization, we investigated the proportion of PPTg/LDTg cholinergic, glutamatergic and GABAergic neurons projecting to the VTA. The retrograde tract tracer Fluoro-Gold (FG) was delivered into the VTA and detected by immunohistochemistry. The neuronal phenotype of retrogradely labeled FG neurons was established by investigating cellular co-expression of FG and transcripts encoding choline acetyltransferase (ChAT; cholinergic marker), glutamic acid decarboxylase (GAD; GABAergic marker) or the vesicular glutamate transporter 2 (vGluT2; glutamatergic marker). Within the PPTg, only 20% (rostral) to 12% (caudal) of the FG neurons co-expressed ChAT mRNA, and 30% (rostral) to 6% (caudal) co-expressed GAD mRNA. In contrast, the majority of FG neurons co-expressed vGluT2 mRNA from the rostral (53%) to caudal (82%) levels. These data indicate that the major input from the PPTg to the VTA is glutamatergic rather than cholinergic. With regard to the LDTg, the FG neurons expressing ChAT mRNA showed a gradual rostro-caudal increase (13% to 28%, respectively), as opposed to the gradual rostro-caudal decrease in FG neurons expressing GAD mRNA (62% to 38%, rostro-caudal distribution). In contrast to the uneven rostro-caudal distribution of both FG/ChAT and FG/GAD neurons in the LDTg, the rostro-caudal distribution of FG neurons expressing vGluT2 mRNA was relatively uniform throughout the LDTg (44% to 50%, rostro-caudal distribution). These data indicated that the major input from the LDTg to the VTA is not cholinergic, but rather glutamatergic or GABAergic. In summary, our results showed that (1) glutamatergic, cholinergic and GABAergic neurons from both the PPTg and the LDTg differentially innervate the VTA, (2) the major input from the PPTg to the VTA is from glutamatergic neurons, and not from cholinergic neurons, (3) the major input from the LDTg to the VTA is not from cholinergic neurons, but from either glutamatergic or GABAergic neurons. In conclusion, despite the fact that glutamatergic, cholinergic and GABAergic neurons from both the PPTg and the LDTg innervate the VTA, these two nuclei are likely to have differential effects on VTA neurotransmission, as the proportion in which their cholinergic, glutamatergic and GABAergic afferents target the VTA is distinct. Project 2. Recent recordings from genetically identified serotonin neurons have provided evidence for neuronal activation of some Dorsal Raphe (DR) serotonin neurons in response to cues predicting reward or reward consumption. In addition, behavioral studies have shown that photoactivation of DR serotonin neurons reinforces instrumental behavior. However, other studies have reported that photoactivation of DR serotonin neurons do not reinforce behavior. Because global serotonin brain manipulations can alter multiple serotonergic projections and transduction pathways as well as multiple components of a given aspect of reinforcement, the role of serotonin on reward function may be better understood by studying the contribution of specific serotonergic pathways. In this regard, DR serotonin neurons heavily innervate the ventral tegmental area (VTA), origin of the mesolimbic dopamine system, a network of known importance for reward and motivational function. Immuno ultrastructural studies have demonstrated that DR serotonin neurons establish synaptic contacts on VTA dopamine neurons, and pharmacological and electrophysiological studies have shown that serotonin is capable of inhibiting or exciting VTA dopamine neurons. The mixed effects of serotonin on VTA dopamine neurons is likely to reflect activation of multiple serotonin receptor subtypes, some of which excite or inhibit dopamine neurons. Nevertheless, a role of VTA in serotonin-mediated reward was initially proposed from studies showing that infusion of serotonin into the VTA potentiates medial forebrain bundle electrical self-stimulation. we examined the ultrastructural and molecular characteristics of the synaptic connectivity between DR serotonin neurons and VTA dopamine neurons, and determined the role of these synapses in behavior. Our results demonstrate that axon terminals from DR serotonin neurons establish symmetric or asymmetric synapses on dopamine neurons of the VTA. Surprisingly, we found that all axon terminals from DR serotonin terminals making asymmetric (putative excitatory) synapses on VTA dopamine neurons co-express vesicular glutamate transporter 3 (VGluT3). Activation of DR serotonin terminals within the VTA elicits a serotonin receptor-mediated excitation of dopamine neurons, and the release of serotonin and glutamate from these terminals induces release of dopamine in nucleus accumbens (nAcc) and promotes conditioned place preference (CPP). Because we have previously demonstrated nAcc dopamine release and a robust reinforcing effect by VTA glutamate release from DR VGluT3-fibers, we compared these effects with those mediated by DR serotonin-fibers. We found that the amount of dopamine release and CPP induced by VTA activation of serotonin-fibers are lower than those induced by activation of VGluT3-fibers. However, CPP induced by activation of SERT-fibers is more resistant to extinction when compared to the CPP induced by activation of VGluT3-fibers.